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Citation for published version: Sherwood, V, Recino, A, Jeffries, A, Ward, A & Chalmers, AD 2010, 'The N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation', Biochemical Journal, vol. 425, no. 2, pp. 303-311. https://doi.org/10.1042/BJ20091318

DOI: 10.1042/BJ20091318

Publication date: 2010

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Download date: 12. May. 2019 The N-terminal RASSF family; A new group of Ras association domain containing proteins, with emerging links to cancer formation.

Victoria Sherwood *†, Asha Recino*, Alex Jeffries*, Andrew Ward*, Andrew D Chalmers*1.

*, Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.

†, Present address, Cell and Experimental Pathology, Lund University, Malmö University Hospital, S-205 02 MALMÖ, Sweden.

1, to whom correspondence should be addressed (e mail [email protected]).

Running title: The N-terminal RASSF family and cancer.

Key words: RASSF7, RASSF8, RASSF9 and RASSF10, ubiquitin fold, tumour suppressor.

Abbreviations used: AP1, activator protein-1; C1, Protein kinase C conserved region; COSMIC, Catalogue of Somatic Mutation in Cancer database; C-terminal, carboxy terminal; DAAX, death-domain-associated protein; DAG, diacylglycerol/phorbol ester binding; ERM, Ezrin, Radixin, moesin; JNK, c-Jun-NH2- kinase; NDR, nuclear Dbf2-related; N-terminal, Amino terminal; PAM, peptidylglycine alpha-amidating monooxygenase; P-CIP1, PAM C-terminal interactor 1; RA, RalGDS/AF6 Ras association; RB, Ras binding domain; RASSF, The Ras- association domain family.

Abstract

The Ras-association domain family (RASSF) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. This review will summarise what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins to the classical RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members, or imposters in the RASSF family.

Introduction Ras proto-oncogenes form part of a superfamily of small GTPases comprising of five families; Ras, Rho, Rab, Ran and Arf [1]. They play a pivotal role in a myriad of cellular processes, including cell growth, apoptosis, adhesion, migration and differentiation [2, 3]. Unsurprisingly then, defects in Ras signalling can result in disease progression, in particular oncogenesis. Indeed, Ras mutations resulting in signalling aberrations, frequently occur in human tumours, particularly in pancreatic and lung adenocarcinomas (Catalogue of Somatic Mutation in Cancer database (Cosmic) [http://www.sanger.ac.uk/genetics/CGP/cosmic/]). Ras proteins carry out their diverse functions by binding to a broad range of Ras effectors and blocking these interactions has been highlighted as an important therapeutic opportunity that could be exploited for cancer treatments [4]. However this requires a better understanding of the effector pathways utilised by Ras [4].

Each Ras effector contains one of a number of Ras binding domains, as example is the RalGDS/AF6 Ras association (RA) domain. This conserved domain is the defining feature of members of the Ras association domain family (RASSF). The family now contains 10 members (RASSF1-10) which are split into two groups, the classical (RASSF1-6) and the amino terminal (N-terminal) RASSF proteins (RASSF7-10) [5]. Members of the classical RASSF proteins have been implicated in a range of biological processes, including the regulation of cell death, cell cycle control and microtubule stability, and are generally regarded as tumour suppressors. This has prompted great interest in these proteins and there are excellent reviews which mainly focus on the classical RASSF family [6-8] and in particular, RASSF1A [9-11]. Recently, four other proteins have been added to the family [5] and renamed RASSF7-10 (Table 1). These N-terminal RASSF proteins represent a new group of potential Ras effectors which may have important biological functions, some of which could well be distinct from previously studied Ras effectors. They may also have a role in cancer progression. In this review we will focus on the N-terminal RASSF proteins. We will summarise what is known about this newly described group of

2 proteins and ask is there any evidence to suggest a role for these proteins in cancer formation? We will also address the question of whether they should be considered as long lost members or imposters in the RASSF family.

RASSF proteins are defined by the presence of a Ras association domain/ ubiquitin fold The defining feature of the RASSF proteins is the presence of a RA domain. This domain was identified by comparing sequences from different Ras binding proteins [12] and is present in over 50 human proteins (SMART database: http://smart.embl- heidelberg.de/). However, the RA nomenclature is potentially misleading as it implies that a protein with this domain will bind Ras. In fact, the binding affinities of RA domains for members of the Ras family show a huge variation and not all will bind Ras [13, 14]. A good example of a RA domain which does not bind Ras is found in the class IX myosin protein, myr 5 [15]. All RA domains are believed to form a similar three dimensional structure called a ubiquitin fold [16], however the RA domain in myr 5 lacks positively charged amino acids which are required for Ras binding [15]. It is not surprising that only a subset of RA domains bind Ras, as the sequences of different RA domains are highly divergent [12]. There are also other ubiquitin fold containing proteins, such as FERM domain containing proteins and ubiquitin, which do not interact with Ras [16]. Another possible cause of confusion is the fact that other Ras effectors such as Raf and PI3K interact with Ras through a domain called a Ras binding (RB) domain. Despite the difference in nomenclature this domain also forms a ubiquitin fold [16]. Thus, Raf, PI3K, RASSF proteins, FERM domain proteins and ubiquitin all share a common structural domain and can be considered part of a ubiquitin fold family [13]. The variation in ability to bind Ras means that a key step in studying RA/ubiquitin fold proteins, such as the RASSF family members, is to establish if the proteins function as Ras effectors, something which will be discussed below.

The classical and N-terminal RASSF proteins have different domain architectures The RA domain/ubiquitin fold of classical RASSF members is found near the carboxy (C)-terminal of the protein, adjacent to a protein-protein interaction domain called the SARAH domain (Fig. 1). This domain is named after the 3 types of proteins that contain it; Salvador (WW45 in vertebrates), RASSF and Hippo (MST1/2 in vertebrates) [17]. SARAH domains have two α-helices which form a novel dimeric anti-parallel helix [18]. Dimerisation between SARAH domains allows Salvador, RASSF and Hippo to form homo and heterodimers. RASSF1 and 5 also contain a diacylglycerol/phorbol ester binding (DAG) domain (Fig. 1), known as protein kinase C conserved region (C1). In RASSF5/Nore1 the C1 domain can form an intramolecular complex with the RA domain/ubiquitin fold and when free bind the lipid phosphatidylinositol 3-phosphate[19].

The N-terminal RASSF proteins have a different domain architecture to the classical RASSFs (Fig. 1A). The RA domain/ubiquitin fold of the N-terminal members is located at the opposite end to the C-terminal location found in the classical RASSF proteins. The RA domains/ubiquitin folds of the two groups also have quite different sequences which form phylogenetically distinct groups (Fig. 2). In addition to the differences in RA domains/ubiquitin folds the N-terminal RASSF members lack an identifiable SARAH motif [5, 17]. However, some caution may be required on this

3 point. The SMART database predicts that RASSF7, 8 and 10 have extensive regions of coiled coil and like SARAH domains, coiled coils can form dimers mediated by hydrophobic residues [20]. Structural studies are required to confirm there is no similarity between the coiled coils of the N-terminal RASSF proteins and SARAH domains of the classical proteins.

RASSF7, 8 and 10 are all located close to members of the Ras family in the genome [5, 21, 22]. This suggests that the N-terminal RASSF proteins may have co-evolved with members of the Ras family. We have not found a similar association between the classical RASSFs and members of the Ras family, so this unusual juxtaposition of a Ras gene and a potential Ras effector represents another distinction between the two groups. The separation between N-terminal and C-terminal RASSF genes is not a recent event, Drosophila and C elegans have both classical (dmRASSF [23] and T24F1.3 [24]) and N-terminal RASSF (Table 1 and [5]) homologues. The differences between classical and N-terminal RASSF proteins prompted us to suggest they are distinct families, with the N-terminal RASSF proteins representing a new group of RA domain/ubiquitin fold containing proteins [5].

Classical RASSF proteins act as tumour suppressors The focus of this review is the N-terminal RASSF proteins, however, before covering these proteins in detail we will summarise what is known about the six classical RASSF (RASSF1-6) proteins. This is not intended to replace comprehensive reviews of the family [6-8], but to allow a comparison with the N-terminal RASSF members.

RASSF1 was originally identified in a yeast-2-hybrid screen and the gene was found to reside at chromosome 3P21.3 [25], a region long suspected to contain at least one tumour suppressor [26]. The expression of one of the RASSF1 transcripts, RASSF1A, was found to be repressed by promoter hypermethylation in lung tumours [25]. Subsequent studies found that RASSF1A was inactivated by methylation in a wide range of tumours (e.g. [27]) and it quickly emerged that RASSF1A is one of the most frequently methylated genes in cancer [9-11]. Restoring expression of RASSF1A reduces tumour growth and knocking out RASSF1A in mice causes an increased frequency of tumour formation [28, 29]. These studies provide convincing evidence that RASSF1A is a tumour suppressor, which is inactivated in a wide range of cancers. Inactivation by promoter methylation occurs with several other members of the classical RASSF family in human tumour cells, including RASSF2, 4 and 5 [6-8], suggesting that they may also be tumour suppressors. Understanding why RASSF1A and other members of the classical RASSF family act as tumour suppressors has been far from straight forward, due to the variety of biological roles they possess. Classical RASSF proteins have been linked to a range of processes, particularly the regulation of apoptosis, cell cycle progression and microtubule stability.

Classical RASSF proteins are key regulators of apoptosis RASSF family members have been linked to promoting apoptosis through a number of effectors [6-8]. One group of effectors are the proapoptotic kinases MST1 and MST2, which bind members of the classical RASSF family [24, 30, 31]. Hippo, the Drosophila homologue of MST1, forms part of an important tumour suppressor network which is crucial for growth control [32, 33]. Hippo functions by regulating the kinase Warts which in turn regulates the transcriptional activator Yorkie, which controls apoptosis-associated genes. Recent work shows that RASSF1A induced

4 apoptosis acts via a similar pathway, which involves MST2 activating LATS, causing the release of YAP which promotes transcription of p73 [34]. NDR (Nuclear Dbf2- related) kinases, which are related to LATS kinases, can also function down stream of MST1 to promote apoptosis [35]. In addition to MST1/2, classical RASSF proteins bind another positive regulator of apoptosis, MOAP1 [36-38]. After death receptor signalling, MOAP-1 and RASSF1A are recruited to the death receptor where the interaction of RASSF1A with MOAP-1 allows MOAP-1 to activate Bax and promote apoptosis [36, 39].

Classical RASSF proteins are important for microtubule stability and cell cycle progression A second function of the classical RASSF proteins is to regulate the cell cycle. Expression of RASSF1A blocks cell cycle progression at a number of stages, including G1, G2-M and in prometaphase [40-43]. The RASSF1A induced arrest in G1 is associated with reduced activity of c-Jun-NH2-kinase (JNK) [44] and activator protein -1 (AP1) [45], which both promote cell cycle progression. RASSF1A also up regulates expression of the cyclin dependant kinase inhibitor p21Cip1/Waf1[46]. These effects are likely to be mediated by a number of effectors. RASSF1A can bind MDM2 and death-domain-associated protein (DAAX). This prevents degradation of the tumour suppressor p53, which would allow p53 to promote cell cycle arrest [47]. RASSF1A can also bind and increase the activity of p120 E4F, a transcriptional repressor of Cyclin A2 [48, 49].

The RASSF1A induced arrest in mitosis is tightly associated with the ability of RASSF1A to associate with microtubules. RASSF1A can bind and stabilise microtubules [41, 43, 50, 51], probably via interacting with a number of microtubule associated proteins [50, 52]. Once bound to microtubule associated proteins RASSF1A appears to function as a scaffold, recruiting multiple regulators of mitosis. Current data suggests these may include Mst1/2, Cdc20, Aurora-A and Ran. Mst1 could signal through the Hippo pathway (see above) to regulate mitotic progression [53]. RASSF1A can also bind and inhibit Cdc20, which activates the anaphase promoting complex [54], although it should be noted that this interaction is controversial [55]. RASSF1A is phosphorylated by the mitotic kinase, Aurora-A [56], and can regulate the activity of Aurora-A [57]. Finally it has recently been shown that Ran can act as a RASSF1A effector to regulate microtubule stability [58]. In addition to mitosis RASSF1A has been linked to cell migration, which is consistent with a role in regulating microtubules [59].

Other members of the classical RASSF family have been linked to cell cycle progression. An example is RASSF5/Nore1, which shows striking similarities to RASSF1A. RASSF5/Nore1 can associate with microtubules [60] and suppress growth by a mechanism which involves p53 activating the expression of p21Cip1 [61]. In summary, classical RASSF proteins have been linked to apoptosis, cell cycle control and the regulation of microtubule stability, all of which may contribute to the tumour suppressor function of these proteins.

Classical RASSF proteins and Ras The presence of a RA domain/ubiquitin fold suggests the classical RASSF proteins will act as Ras effectors. However, as discussed above, not all RA domains bind Ras, and for many of the classical RASSF proteins it is not clear if they function as Ras

5 effectors to mediate the processes described above. RASSF5/Nore1 is perhaps the best documented Ras effector of the RASSF family. The splice variant RASSF5A/Nore1A was identified as a Ras interacting protein by yeast-two-hybrid and the endogenous protein interacts with Ras following addition of EGF [62]. RASSF5A/Nore1A is also the first member of the RASSF family to have the crystal structure of its RA domain/ubiquitin fold determined [63]. This was carried out in complex with Ras and demonstrated that the region which interacts with Ras is extended compared with other Ras effectors. This lengthened interface provides the RASSF5A/Nore1A – Ras complex with a prolonged lifetime compared to other Ras effectors. However, a physiological role for a RASSF5A/Nore1A-Ras complex is yet to be identified. The splice variant RASSF5B/Nore1B (also known as RAPL) is a Ras effector with a well documented physiological role in T cell signalling, where it associates with the Ras protein Rap1 [64].

RASSF1A can bind Ras in a GTP dependant manner [65], but it binds with a much lower affinity than RASSF5/Nore1 [63, 66]. This raises the question, is RASSF1A a genuine Ras effector? An endogenous RASSF1A-Ras complex has been described [67], but similar to the RASSF5A/Nore1A-Ras complex, the physiological role of this complex is not known. A recent twist to this story is that RASSF1A can bind to the small GTPase Ran [58] and regulate microtubule organisation (discussed above). The binding appears direct and can be seen with endogenous protein. Ran is not a member of the Ras family but is part of the large superfamily of related small GTPases [1]. This suggests that RASSF1A functions by binding Ran in addition to, or instead of, binding Ras. It also raises the possibility that RASSF1A and other RASSF proteins might bind other small GTPases in addition to Ras and Ran. Future work is required to untangle the biology of the classical RASSF proteins and the role that Ras and other small GTPases play in their function.

The N-terminal RASSF family The difference in domain architecture and sequence of the RA domains prompted us to propose that the N-terminal RASSF proteins are a distinct family from the classical RASSF proteins [5]. This makes the decision to add them to the RASSF family look questionable. However, one crucial benefit of the renaming is to group the N-terminal RASSF proteins together for the first time. This makes it possible to compare what is known about each member. To achieve this we have searched the literature for work relating to each N-terminal RASSF protein. We used the 11 different names which have been given to the vertebrate members (Table 1) and it is important to point out that many of the references cited here use the older nomenclature. Given the importance of the work on the Drosophila classical RASSF protein [23], we have also looked at the three potential N-terminal Drosophila members. In the following sections we will summarise what is known about each of the N-terminal RASSF proteins and where appropriate relate it back to what is known about the classical RASSF proteins.

RASSF7: The first RASSF protein to be described? RASSF7 was originally identified by a study which set out to sequence genes which are located close to HRas in the genome [21]. The authors found an unstudied gene and called it Hras1 cluster 1, HRC-1. HRC-1 was recently renamed RASSF7, presumably because the protein it encodes contains an RA domain/ubiquitin fold and was not part of a recognised family. The hypothesis of the authors who identified

6 RASSF7/HRC-1 was that it might be a growth regulator because it was close to HRas in the genome [21]. They also suggested, based on Southern blotting, that RASSF7/HRC-1 might be part of a large family of related proteins. This was six years before RASSF5/Nore1 was identified as a potential Ras effector [62] and it is only recently that the authors predictions about RASSF7 have begun to be confirmed.

The genomic position of RASSF7 places it in close proximity to the HRAS1 minisatellite which is immediately down stream of HRas. Rare alleles of this minisatellite were shown to be associated with cancer risk [68, 69] and it was proposed that altered expression of RASSF7 might contribute to the increased risk [70]. This generated a great deal of interest in the region, however subsequent studies using improved technology failed to find a link [71, 72] and the idea that rare alleles of the minisatellite are associated with cancer risk has fallen from favour.

RASSF7: a hypoxic response gene which is up regulated in certain cancers. The advent of genomic screening technology has made it possible to interrogate the entire genome for genes which are misregulated in cancer. Several microarray studies have shown that RASSF7 is up regulated in cancer (Table 2). One example is in pancreatic cancer. Two independent studies have found that RASSF7 expression is increased in pancreatic ductal adenocarcinoma relative to normal tissue [73-75]. In addition to ductal adenocarcinoma, RASSF7 has increased expression in a second type of pancreatic cancer, islet cell tumours [76]. This study selected RASSF7 as a key gene whose expression can be used to identify islet cell tumours. RASSF7 expression is also increased in endometrial cancer [77]. Similar to the situation in islet cell tumours, RASSF7 showed a large increase in expression and was selected as one of the top 50 genes which distinguish malignant from normal endometrium. Finally RASSF7 lies in a genomic region which is amplified in ovarian clear cell carcinoma and its expression is increased in these cancers, correlating with the genomic amplification [78].

Interestingly, recent work offers plausible explanations as to why RASSF7 may be up regulated in cancer samples. Hypoxia, which occurs in solid tumours, is known to cause a large number of gene expression changes [79] and RASSF7 expression was found to be up regulated by hypoxia in MCF7 breast cancer cells [80] and in human umbilical vein endothelial cells [81]. This predicts that the hypoxic environment found in solid tumours would cause an increase in RASSF7 expression. Furthermore, RASSF7 is also down-regulated by the tumour suppressor, BRCA1, suggesting its expression would be increased in cancer cells which have lost BRCA1 function [82].

RASSF7 is required for cell death and proliferation An important question is what role RASSF7 plays in these cancerous cells? There is currently no evidence to suggest that increased expression of RASSF7 promotes cancer formation. However, RASSF7 function has been linked to some key biological processes including the regulation of cell death and proliferation. RASSF7 has been shown to be required for necroptosis [83], a regulated form of necrosis which is distinct from apoptosis. A large scale siRNA screen was carried out to find proteins required for necroptosis and this identified RASSF7 and RASSF8.

We identified Xenopus RASSF7 in a microarray screen [84] and subsequently found that in Xenopus, RASSF7 is essential for cell cycle progression and cell survival [5].

7 In cells where RASSF7 is knocked-down, mitotic spindles fail to form and cells arrest in mitosis. This causes nuclear fragmentation and apoptosis. Consistent with a role in mitotic progression, Xenopus RASSF7 is localised at the centrosome. However, Xenopus RASSF7 is not a core component of the centrosome, rather it appears to be enriched at the centrosome because it interacts with the minus ends of microtubules. Preliminary data from a large scale screen suggests that the Drosophila homologue may also be required for cell proliferation. Knockdown by RNA interference caused a reduction in the mitotic index and weak spindle defects [85].

RASSF7, like other RASSF proteins, contains no catalytic domain so to understand its function it is crucial to identify the proteins it interacts with. Yeast-2-hybrid studies have identified potential binding partners for human RASSF7. These include CHMP1B, which is associated with endosomal membrane trafficking, and DISC1 which interestingly interacts with microtubules [86, 87].

RASSF8 is located in a genomic region associated with lung cancer risk The sequence for RASSF8 was first deposited into the NCBI data base by Hoon and Yuzuki in 1996 (accession number Q8NHQ8) and called ‘human carcinoma associated HoJ-1’. However, there appears to have been no publication associated with this submission. Subsequently, RASSF8 was characterised as a gene involved in a chromosomal translocation associated with a complex type of synpolydactyly [88, 89]. RASSF8 was found to be located on chromosome 12 and referred to as C12orf2. The chromosomal translocation fused RASSF8 with the fibulin-1 gene (22q13.3). It is believed that this disrupts a fibulin-1 splice variant, causing the synpolydactyly. C12orf2 was then renamed RASSF8, presumably because it contains a RA domain/ubiquitin fold and was not part of a recognised family. This occurred at the same time as HRC-1 was renamed RASSF7 and both proteins were added to the RASSF family.

RASSF8 is located about 700 Kb from the KRas2 gene [22] and both genes lie in a region called Pals1. This region has been identified as a major susceptibility locus in a mouse model for lung carcinogenesis [90]. There are a number of genes in this region but it is mutations in the KRas2 gene that are believed to be responsible for the increased risk [91]. The homologous region in humans has also been associated with increased lung adenocarcinoma risk [92]. However, in humans it is not clear if it is KRas2 that is responsible. Analysis of the region in a Japanese population, identified the D12S1034 microsatellite as being most tightly associated with lung cancer risk [93]. The D12S1034 locus showed a bigger difference between cases and controls than the microsatellite adjacent to KRas2. This argues that in some human cancers, susceptibility may be due to a mutation in a gene adjacent to D12S1034 rather than in KRas2 itself. RASSF8 lies within 20 Kb of D12S1034 making it a good candidate gene, particularly as RASSF8 has also been described as a potential tumour suppressor in lung cancer (see below). However, common polymorphisms in RASSF8 are not associated with cancer risk in an Italian population [94]. Thus, currently it is not clear if there is the link between RASSF8 and the increased lung cancer risk associated with the Pals1 region.

RASSF8 is a potential tumour suppressor There are several lines of evidence to suggest that RASSF8 may be a tumour suppressor (altered expression levels are summarised in Table 2). The best

8 characterised example is in lung cancer [22]. In lung adenocarcinoma RASSF8 transcript levels were reduced compared with normal tissue. Over-expression of RASSF8 protein in lung cancer cell lines also inhibited anchorage-independent growth, which has been correlated with tumour progression and metastasis. In addition to lung adenocarcinoma, RASSF8 expression is also down-regulated in male germ cell tumours [95], despite the fact that the gene lies in a genomic region which shows gain in almost 100% of these cancers. Finally, RASSF8 was identified as a candidate gene involved in leukaemia and lymphoma formation in a study on retroviral-induced blood cancers in mice [96]. This model assumes that oncogenes and tumour suppressors often lie near common retroviral insertion sites. A genomic region next to RASSF8 was targeted seven times, making it one of the most frequently hit sites in the study. This suggests that mis-regulation of RASSF8 may contribute to leukaemia and lymphoma formation, so it is interesting that RASSF8 has higher expression in human hematopoietic stem cells and is required for blood cell development in Zebrafish [97]. It is not known why RASSF8 might be a tumour suppressor but it is interesting that, similar to RASSF7, it is required for cell death by necroptosis [83]. This form of cell death may be particularly important in cells with deficiencies in their apoptotic machinery, such as tumour cells, so a role in necroptosis would be consistent with a tumour suppressor function.

Mass spectrometry and yeast-2-hybrid screens have identified a number of potential binding partners for RASSF8; including the scaffolding protein, 14-3-3γ which binds phosphoproteins to modulate their function [98]; and FRMD4, an ERM (Ezrin, Radixin, Moesin) protein that links membrane domains to actin and PSMD4, a component of the proteosome [99]. These potential binding partners offer interesting leads for future work aimed at understanding why RASSF8 may function as a tumour suppressor.

RASSF9 is a Ras binding protein that has been linked to vesicle trafficking RASSF9 was first identified by a yeast-2-hybrid screen as a protein that interacted with the cytoplasmic domain of peptidylglycine alpha-amidating monooxygenase (PAM) [100]. Based on this interaction it was named PAM C-terminal interactor 1 (P- CIP1). PAM is a transmembrane protein found in secretory vesicles of neurons and endocrine cells, where it catalyses the α-amidation of bioactive peptides such as oxytocin and vasopressin. This modification is essential for the activity of these peptides [101]. We realised that PCIP-1 contains an RA domain/ubiquitin fold and is closely related to RASSF7+8 and suggested it should be renamed RASSF9 [5]. The binding of RASSF9/P-CIP1 to PAM was confirmed and RASSF9/P-CIP1 was found to associate with recycling endosomes [102]. This led to the model that it might bind the cytoplasmic domain of PAM during recycling of the enzyme [102], an interaction that may be regulated by phosphorylation as the cytoplasmic domain of PAM is known to be multiply phosphorylated [103]. However, RASSF9 mRNA is expressed much more widely than PAM, so RASSF9 might have additional roles, perhaps binding other transmembrane proteins.

Interestingly RASSF9 is the one member of the N-terminal RASSF proteins which has been shown to bind RAS proteins. Pull down experiments with RASSF9 and Ras family GTPases showed that RASSF9 binds N-Ras, K-Ras and R-Ras [14]. An issue that remains to be addressed is whether RASSF9 binds endogenous Ras, or other

9 small GTPases, something which has not been straightforward to answer for the classical RASSF proteins (see above).

RASSF10 is a candidate tumour suppressor in childhood leukaemia We discovered that a predicted protein was similar in sequence to RASSF9 and named this protein RASSF10 [5]. This gene was completely unstudied, until recent work showing that it is a candidate tumour suppressor in childhood leukaemias [104]. The transcript of RASSF10 was characterised and found to be shorter than the predicted version. The protein encoded by the shorter version is more similar to RASSF7-9 and is used in Figure 1. RASSF10 contains a large CpG island and given the frequent inactivation of classical RASSFs by promoter hyper methylation (see above) the authors examined the methylation status of this gene in childhood leukaemia. They found that RASSF10 was frequently methylated in leukaemia cell lines (100%) and T cell acute lymphocytic leukaemias (T-All) (88%), but not in normal bone marrow and blood samples. RASSF10 was also rarely methylated in B- ALL (16%). Inhibiting this methylation caused an upregulation of expression in the leukaemia cell lines. This data strongly suggests that RASSF10 expression is inhibited by promoter methylation in a high percentage of T-All, raising the possibility that RASSF10 might function as a tumour suppressor in these cancers. ESTs for RASSF10 are present in a number of tissues [104] and it will be interesting to see if RASSF10 is methylated in tumours derived from these tissues.

The function of RASSF10 remains unstudied in vertebrates. There is a potential Drosophila homologue (Table 1). However little is known about this gene except that it is expressed in precursors of the peripheral nervous system [105], and knocking down its function impairs Hedgehog signalling [106]. RASSF10 offers exciting opportunities for future study.

Concluding remarks The N-terminal RASSF proteins have a different domain architecture from the classical RASSF proteins and so we proposed that they should be considered as a separate family [5]. Donninger and colleagues came to a similar conclusion for RASSF7 and 8, suggesting that they are a separate sub-family distinct from the ‘true’ RASSF proteins [10]. If the N-terminal and classical RASSF proteins are members of different families then one might expect that there will be little overlap between their biology. However, RASSF7 and RASSF1A show similar centrosomal localisation and mitotic defects when knocked down and RASSF10 and members of the classical RASSF family both show promoter hypermethylation. These similarities might suggest that the N-terminal RASSF proteins are genuine RASSF proteins. However, we feel the differences between them still outweigh the similarities and that the N- terminal RASSF proteins are not ‘true’ RASSF proteins, but a separate family. Emerging evidence presented in this review suggests that the N-terminal RASSF proteins might play a role in tumour formation (summarised in Fig. 3). There is now an exciting opportunity to study this new group of proteins in more detail and confirm whether they are important in oncogenic progression.

Acknowledgements We apologise to those whose work we could not cite due to space restrictions. Dr Eric O’Neill and Dr Paul Whitley are thanked for critical comments on the manuscript.

Funding AR, Marie Curie PhD studentship [MCEST-CT-2005-019822]. AW, Medical Research Council Cooperative Group Grant (66812). AC, MRC Fellowship [G120/844] and Cancer Research UK Grant [C26932/A9548].

References 1 Takai, Y., Sasaki, T. and Matozaki, T. (2001) Small GTP-binding proteins. Physiol Rev. 81, 153-208 2 Cully, M. and Downward, J. (2008) SnapShot: Ras Signaling. Cell. 133, 1292- 1292 e1291 3 Hancock, J. F. and Parton, R. G. (2005) Ras plasma membrane signalling platforms. Biochem J. 389, 1-11 4 Tanaka, T. and Rabbitts, T. H. (2008) Interfering with protein-protein interactions: potential for cancer therapy. Cell Cycle. 7, 1569-1574 5 Sherwood, V., Manbodh, R., Sheppard, C. and Chalmers, A. D. (2008) RASSF7 Is a Member of a New Family of RAS Association Domain-containing Proteins and Is Required for Completing Mitosis. Mol Biol Cell. 19, 1772-1782 6 Avruch, J., Xavier, R., Bardeesy, N., Zhang, X. F., Praskova, M., Zhou, D. and Xia, F. (2009) Rassf family of tumor suppressor polypeptides. J Biol Chem. 284, 11001-11005 7 Richter, A. M., Pfeifer, G. P. and Dammann, R. H. (2009) The RASSF proteins in cancer; from epigenetic silencing to functional characterization. Biochim Biophys Acta 8 van der Weyden, L. and Adams, D. J. (2007) The Ras-association domain family (RASSF) members and their role in human tumourigenesis. Biochim Biophys Acta. 1776, 58-85 9 Dammann, R., Schagdarsurengin, U., Seidel, C., Strunnikova, M., Rastetter, M., Baier, K. and Pfeifer, G. P. (2005) The tumor suppressor RASSF1A in human carcinogenesis: an update. Histol Histopathol. 20, 645-663 10 Donninger, H., Vos, M. D. and Clark, G. J. (2007) The RASSF1A tumor suppressor. J Cell Sci. 120, 3163-3172 11 Hesson, L. B., Cooper, W. N. and Latif, F. (2007) The role of RASSF1A methylation in cancer. Dis Markers. 23, 73-87 12 Ponting, C. P. and Benjamin, D. R. (1996) A novel family of Ras-binding domains. Trends Biochem Sci. 21, 422-425 13 Wohlgemuth, S., Kiel, C., Kramer, A., Serrano, L., Wittinghofer, F. and Herrmann, C. (2005) Recognizing and defining true Ras binding domains I: biochemical analysis. J Mol Biol. 348, 741-758 14 Rodriguez-Viciana, P., Sabatier, C. and McCormick, F. (2004) Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol Cell Biol. 24, 4943-4954 15 Kalhammer, G., Bahler, M., Schmitz, F., Jockel, J. and Block, C. (1997) Ras- binding domains: predicting function versus folding. FEBS Lett. 414, 599-602 16 Herrmann, C. (2003) Ras-effector interactions: after one decade. Curr Opin Struct Biol. 13, 122-129 17 Scheel, H. and Hofmann, K. (2003) A novel interaction motif, SARAH, connects three classes of tumor suppressor. Curr Biol. 13, R899-900

11 18 Hwang, E., Ryu, K. S., Paakkonen, K., Guntert, P., Cheong, H. K., Lim, D. S., Lee, J. O., Jeon, Y. H. and Cheong, C. (2007) Structural insight into dimeric interaction of the SARAH domains from Mst1 and RASSF family proteins in the apoptosis pathway. Proc Natl Acad Sci U S A. 104, 9236-9241 19 Harjes, E., Harjes, S., Wohlgemuth, S., Muller, K. H., Krieger, E., Herrmann, C. and Bayer, P. (2006) GTP-Ras disrupts the intramolecular complex of C1 and RA domains of Nore1. Structure. 14, 881-888 20 Grigoryan, G. and Keating, A. E. (2008) Structural specificity in coiled-coil interactions. Curr Opin Struct Biol. 18, 477-483 21 Weitzel, J. N., Kasperczyk, A., Mohan, C. and Krontiris, T. G. (1992) The HRAS1 gene cluster: two upstream regions recognizing transcripts and a third encoding a gene with a leucine zipper domain. Genomics. 14, 309-319 22 Falvella, F. S., Manenti, G., Spinola, M., Pignatiello, C., Conti, B., Pastorino, U. and Dragani, T. A. (2006) Identification of RASSF8 as a candidate lung tumor suppressor gene. Oncogene. 25, 3934-3938 23 Polesello, C., Huelsmann, S., Brown, N. H. and Tapon, N. (2006) The Drosophila RASSF homolog antagonizes the hippo pathway. Curr Biol. 16, 2459- 2465 24 Khokhlatchev, A., Rabizadeh, S., Xavier, R., Nedwidek, M., Chen, T., Zhang, X. F., Seed, B. and Avruch, J. (2002) Identification of a novel Ras-regulated proapoptotic pathway. Curr Biol. 12, 253-265 25 Dammann, R., Li, C., Yoon, J. H., Chin, P. L., Bates, S. and Pfeifer, G. P. (2000) Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet. 25, 315-319 26 Lerman, M. I. and Minna, J. D. (2000) The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res. 60, 6116- 6133 27 Agathanggelou, A., Honorio, S., Macartney, D. P., Martinez, A., Dallol, A., Rader, J., Fullwood, P., Chauhan, A., Walker, R., Shaw, J. A., Hosoe, S., Lerman, M. I., Minna, J. D., Maher, E. R. and Latif, F. (2001) Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene. 20, 1509-1518 28 van der Weyden, L., Tachibana, K. K., Gonzalez, M. A., Adams, D. J., Ng, B. L., Petty, R., Venkitaraman, A. R., Arends, M. J. and Bradley, A. (2005) The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis. Mol Cell Biol. 25, 8356-8367 29 Tommasi, S., Dammann, R., Zhang, Z., Wang, Y., Liu, L., Tsark, W. M., Wilczynski, S. P., Li, J., You, M. and Pfeifer, G. P. (2005) Tumor susceptibility of Rassf1a knockout mice. Cancer Res. 65, 92-98 30 Praskova, M., Khoklatchev, A., Ortiz-Vega, S. and Avruch, J. (2004) Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras. Biochem J. 381, 453-462 31 Cooper, W. N., Hesson, L. B., Matallanas, D., Dallol, A., von Kriegsheim, A., Ward, R., Kolch, W. and Latif, F. (2009) RASSF2 associates with and stabilizes the proapoptotic kinase MST2. Oncogene. 28, 2988-2998 32 Harvey, K. and Tapon, N. (2007) The Salvador-Warts-Hippo pathway - an emerging tumour-suppressor network. Nat Rev Cancer. 7, 182-191

12 33 Saucedo, L. J. and Edgar, B. A. (2007) Filling out the Hippo pathway. Nat Rev Mol Cell Biol. 8, 613-621 34 Matallanas, D., Romano, D., Yee, K., Meissl, K., Kucerova, L., Piazzolla, D., Baccarini, M., Vass, J. K., Kolch, W. and O'Neill, E. (2007) RASSF1A elicits apoptosis through an MST2 pathway directing proapoptotic transcription by the p73 tumor suppressor protein. Mol Cell. 27, 962-975 35 Vichalkovski, A., Gresko, E., Cornils, H., Hergovich, A., Schmitz, D. and Hemmings, B. A. (2008) NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis. Curr Biol. 18, 1889-1895 36 Baksh, S., Tommasi, S., Fenton, S., Yu, V. C., Martins, L. M., Pfeifer, G. P., Latif, F., Downward, J. and Neel, B. G. (2005) The tumor suppressor RASSF1A and MAP-1 link death receptor signaling to Bax conformational change and cell death. Mol Cell. 18, 637-650 37 Vos, M. D., Dallol, A., Eckfeld, K., Allen, N. P., Donninger, H., Hesson, L. B., Calvisi, D., Latif, F. and Clark, G. J. (2006) The RASSF1A tumor suppressor activates Bax via MOAP-1. J Biol Chem. 281, 4557-4563 38 Allen, N. P., Donninger, H., Vos, M. D., Eckfeld, K., Hesson, L., Gordon, L., Birrer, M. J., Latif, F. and Clark, G. J. (2007) RASSF6 is a novel member of the RASSF family of tumor suppressors. Oncogene. 26, 6203-6211 39 Foley, C. J., Freedman, H., Choo, S. L., Onyskiw, C., Fu, N. Y., Yu, V. C., Tuszynski, J., Pratt, J. C. and Baksh, S. (2008) Dynamics of RASSF1A/MOAP-1 association with death receptors. Mol Cell Biol. 28, 4520-4535 40 Shivakumar, L., Minna, J., Sakamaki, T., Pestell, R. and White, M. A. (2002) The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation. Mol Cell Biol. 22, 4309-4318 41 Liu, L., Tommasi, S., Lee, D. H., Dammann, R. and Pfeifer, G. P. (2003) Control of microtubule stability by the RASSF1A tumor suppressor. Oncogene. 22, 8125-8136 42 Song, M. S., Song, S. J., Ayad, N. G., Chang, J. S., Lee, J. H., Hong, H. K., Lee, H., Choi, N., Kim, J., Kim, H., Kim, J. W., Choi, E. J., Kirschner, M. W. and Lim, D. S. (2004) The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex. Nat Cell Biol. 6, 129-137 43 Rong, R., Jin, W., Zhang, J., Sheikh, M. S. and Huang, Y. (2004) Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest. Oncogene. 23, 8216-8230 44 Whang, Y. M., Kim, Y. H., Kim, J. S. and Yoo, Y. D. (2005) RASSF1A suppresses the c-Jun-NH2-kinase pathway and inhibits cell cycle progression. Cancer Res. 65, 3682-3690 45 Deng, Z. H., Wen, J. F., Li, J. H., Xiao, D. S. and Zhou, J. H. (2008) Activator protein-1 involved in growth inhibition by RASSF1A gene in the human gastric carcinoma cell line SGC7901. World J Gastroenterol. 14, 1437-1443 46 Thaler, S., Hahnel, P. S., Schad, A., Dammann, R. and Schuler, M. (2009) RASSF1A mediates p21Cip1/Waf1-dependent cell cycle arrest and senescence through modulation of the Raf-MEK-ERK pathway and inhibition of Akt. Cancer Res. 69, 1748-1757 47 Song, M. S., Song, S. J., Kim, S. Y., Oh, H. J. and Lim, D. S. (2008) The tumour suppressor RASSF1A promotes MDM2 self-ubiquitination by disrupting the MDM2-DAXX-HAUSP complex. Embo J. 27, 1863-1874 48 Ahmed-Choudhury, J., Agathanggelou, A., Fenton, S. L., Ricketts, C., Clark, G. J., Maher, E. R. and Latif, F. (2005) Transcriptional regulation of cyclin A2 by

13 RASSF1A through the enhanced binding of p120E4F to the cyclin A2 promoter. Cancer Res. 65, 2690-2697 49 Fenton, S. L., Dallol, A., Agathanggelou, A., Hesson, L., Ahmed-Choudhury, J., Baksh, S., Sardet, C., Dammann, R., Minna, J. D., Downward, J., Maher, E. R. and Latif, F. (2004) Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. 64, 102-107 50 Dallol, A., Agathanggelou, A., Fenton, S. L., Ahmed-Choudhury, J., Hesson, L., Vos, M. D., Clark, G. J., Downward, J., Maher, E. R. and Latif, F. (2004) RASSF1A interacts with microtubule-associated proteins and modulates microtubule dynamics. Cancer Res. 64, 4112-4116 51 Vos, M. D., Martinez, A., Elam, C., Dallol, A., Taylor, B. J., Latif, F. and Clark, G. J. (2004) A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability. Cancer Res. 64, 4244-4250 52 Song, M. S., Chang, J. S., Song, S. J., Yang, T. H., Lee, H. and Lim, D. S. (2005) The centrosomal protein RAS association domain family protein 1A (RASSF1A)-binding protein 1 regulates mitotic progression by recruiting RASSF1A to spindle poles. J Biol Chem. 280, 3920-3927 53 Guo, C., Tommasi, S., Liu, L., Yee, J. K., Dammann, R. and Pfeifer, G. P. (2007) RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor-suppressor network. Curr Biol. 17, 700-705 54 Song, M. S. and Lim, D. S. (2004) Control of APC-Cdc20 by the tumor suppressor RASSF1A. Cell Cycle. 3, 574-576 55 Liu, L., Baier, K., Dammann, R. and Pfeifer, G. P. (2007) The tumor suppressor RASSF1A does not interact with Cdc20, an activator of the anaphase- promoting complex. Cell Cycle. 6, 1663-1665 56 Rong, R., Jiang, L. Y., Sheikh, M. S. and Huang, Y. (2007) Mitotic kinase Aurora-A phosphorylates RASSF1A and modulates RASSF1A-mediated microtubule interaction and M-phase cell cycle regulation. Oncogene. 26, 7700-7708 57 Liu, L., Guo, C., Dammann, R., Tommasi, S. and Pfeifer, G. P. (2008) RASSF1A interacts with and activates the mitotic kinase Aurora-A. Oncogene. 27, 6175-6186 58 Dallol, A., Hesson, L. B., Matallanas, D., Cooper, W. N., O'Neill, E., Maher, E. R., Kolch, W. and Latif, F. (2009) RAN GTPase is a RASSF1A effector involved in controlling microtubule organization. Curr Biol. 19, 1227-1232 59 Dallol, A., Agathanggelou, A., Tommasi, S., Pfeifer, G. P., Maher, E. R. and Latif, F. (2005) Involvement of the RASSF1A tumor suppressor gene in controlling cell migration. Cancer Res. 65, 7653-7659 60 Moshnikova, A., Frye, J., Shay, J. W., Minna, J. D. and Khokhlatchev, A. V. (2006) The growth and tumor suppressor NORE1A is a cytoskeletal protein that suppresses growth by inhibition of the ERK pathway. J Biol Chem. 281, 8143-8152 61 Calvisi, D. F., Donninger, H., Vos, M. D., Birrer, M. J., Gordon, L., Leaner, V. and Clark, G. J. (2009) NORE1A tumor suppressor candidate modulates p21CIP1 via p53. Cancer Res. 69, 4629-4637 62 Vavvas, D., Li, X., Avruch, J. and Zhang, X. F. (1998) Identification of Nore1 as a potential Ras effector. J Biol Chem. 273, 5439-5442 63 Stieglitz, B., Bee, C., Schwarz, D., Yildiz, O., Moshnikova, A., Khokhlatchev, A. and Herrmann, C. (2008) Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II. Embo J. 27, 1995-2005

14 64 Kinashi, T. (2007) Integrin regulation of lymphocyte trafficking: lessons from structural and signaling studies. Adv Immunol. 93, 185-227 65 Vos, M. D. and Clark, G. J. (2005) RASSF Family Proteins and Ras Transformation. Methods Enzymol. 407, 311-322 66 Ortiz-Vega, S., Khokhlatchev, A., Nedwidek, M., Zhang, X. F., Dammann, R., Pfeifer, G. P. and Avruch, J. (2002) The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1. Oncogene. 21, 1381-1390 67 Calvisi, D. F., Ladu, S., Gorden, A., Farina, M., Conner, E. A., Lee, J. S., Factor, V. M. and Thorgeirsson, S. S. (2006) Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology. 130, 1117-1128 68 Krontiris, T. G., Devlin, B., Karp, D. D., Robert, N. J. and Risch, N. (1993) An association between the risk of cancer and mutations in the HRAS1 minisatellite locus. N Engl J Med. 329, 517-523 69 Krontiris, T. G., DiMartino, N. A., Colb, M. and Parkinson, D. R. (1985) Unique allelic restriction fragments of the human Ha-ras locus in leukocyte and tumour DNAs of cancer patients. Nature. 313, 369-374 70 Weitzel, J. N., Ding, S., Larson, G. P., Nelson, R. A., Goodman, A., Grendys, E. C., Ball, H. G. and Krontiris, T. G. (2000) The HRAS1 minisatellite locus and risk of ovarian cancer. Cancer Res. 60, 259-261 71 Firgaira, F. A., Seshadri, R., McEvoy, C. R., Dite, G. S., Giles, G. G., McCredie, M. R., Southey, M. C., Venter, D. J. and Hopper, J. L. (1999) HRAS1 rare minisatellite alleles and breast cancer in Australian women under age forty years. J Natl Cancer Inst. 91, 2107-2111 72 Tamimi, R. M., Hankinson, S. E., Ding, S., Gagalang, V., Larson, G. P., Spiegelman, D., Colditz, G. A., Krontiris, T. G. and Hunter, D. J. (2003) The HRAS1 variable number of tandem repeats and risk of breast cancer. Cancer Epidemiol Biomarkers Prev. 12, 1528-1530 73 Brandt, R., Grutzmann, R., Bauer, A., Jesnowski, R., Ringel, J., Lohr, M., Pilarsky, C. and Hoheisel, J. D. (2004) DNA microarray analysis of pancreatic malignancies. Pancreatology. 4, 587-597 74 Friess, H., Ding, J., Kleeff, J., Fenkell, L., Rosinski, J. A., Guweidhi, A., Reidhaar-Olson, J. F., Korc, M., Hammer, J. and Buchler, M. W. (2003) Microarray- based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer. Cell Mol Life Sci. 60, 1180-1199 75 Logsdon, C. D., Simeone, D. M., Binkley, C., Arumugam, T., Greenson, J. K., Giordano, T. J., Misek, D. E., Kuick, R. and Hanash, S. (2003) Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res. 63, 2649-2657 76 Lowe, A. W., Olsen, M., Hao, Y., Lee, S. P., Taek Lee, K., Chen, X., van de Rijn, M. and Brown, P. O. (2007) Gene expression patterns in pancreatic tumors, cells and tissues. PLoS ONE. 2, e323 77 Mutter, G. L., Baak, J. P., Fitzgerald, J. T., Gray, R., Neuberg, D., Kust, G. A., Gentleman, R., Gullans, S. R., Wei, L. J. and Wilcox, M. (2001) Global expression changes of constitutive and hormonally regulated genes during endometrial neoplastic transformation. Gynecol Oncol. 83, 177-185 78 Tan, D. S., Lambros, M. B., Rayter, S., Natrajan, R., Vatcheva, R., Gao, Q., Marchio, C., Geyer, F. C., Savage, K., Parry, S., Fenwick, K., Tamber, N., Mackay, A., Dexter, T., Jameson, C., McCluggage, W. G., Williams, A., Graham, A., Faratian, D., El-Bahrawy, M., Paige, A. J., Gabra, H., Gore, M. E., Zvelebil, M., Lord, C. J.,

15 Kaye, S. B., Ashworth, A. and Reis-Filho, J. S. (2009) PPM1D is a potential therapeutic target in ovarian clear cell carcinomas. Clin Cancer Res. 15, 2269-2280 79 Kenneth, N. S. and Rocha, S. (2008) Regulation of gene expression by hypoxia. Biochem J. 414, 19-29 80 Camps, C., Buffa, F. M., Colella, S., Moore, J., Sotiriou, C., Sheldon, H., Harris, A. L., Gleadle, J. M. and Ragoussis, J. (2008) hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer. Clin Cancer Res. 14, 1340-1348 81 Liang, G. P., Su, Y. Y., Chen, J., Yang, Z. C., Liu, Y. S. and Luo, X. D. (2009) Analysis of the early adaptive response of endothelial cells to hypoxia via a long serial analysis of gene expression. Biochem Biophys Res Commun. 384, 415- 419 82 Welcsh, P. L., Lee, M. K., Gonzalez-Hernandez, R. M., Black, D. J., Mahadevappa, M., Swisher, E. M., Warrington, J. A. and King, M. C. (2002) BRCA1 transcriptionally regulates genes involved in breast tumorigenesis. Proc Natl Acad Sci U S A. 99, 7560-7565 83 Hitomi, J., Christofferson, D. E., Ng, A., Yao, J., Degterev, A., Xavier, R. J. and Yuan, J. (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 135, 1311-1323 84 Chalmers, A. D., Lachani, K., Shin, Y., Sherwood, V., Cho, K. W. and Papalopulu, N. (2006) Grainyhead-like 3, a transcription factor identified in a microarray screen, promotes the specification of the superficial layer of the embryonic epidermis. Mech Dev. 123, 702-718 85 Goshima, G., Wollman, R., Goodwin, S. S., Zhang, N., Scholey, J. M., Vale, R. D. and Stuurman, N. (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science. 316, 417-421 86 Morris, J. A., Kandpal, G., Ma, L. and Austin, C. P. (2003) DISC1 (Disrupted- In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum Mol Genet. 12, 1591-1608 87 Tsang, H. T., Connell, J. W., Brown, S. E., Thompson, A., Reid, E. and Sanderson, C. M. (2006) A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex. Genomics. 88, 333-346 88 Debeer, P., Schoenmakers, E. F., Thoelen, R., Holvoet, M., Kuittinen, T., Fabry, G., Fryns, J. P., Goodman, F. R. and Van de Ven, W. J. (2000) Physical map of a 1.5 mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint. Eur J Hum Genet. 8, 561-570 89 Debeer, P., Schoenmakers, E. F., Twal, W. O., Argraves, W. S., De Smet, L., Fryns, J. P. and Van De Ven, W. J. (2002) The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly. J Med Genet. 39, 98-104 90 Gariboldi, M., Manenti, G., Canzian, F., Falvella, F. S., Radice, M. T., Pierotti, M. A., Della Porta, G., Binelli, G. and Dragani, T. A. (1993) A major susceptibility locus to murine lung carcinogenesis maps on chromosome 6. Nat Genet. 3, 132-136 91 To, M. D., Perez-Losada, J., Mao, J. H., Hsu, J., Jacks, T. and Balmain, A. (2006) A functional switch from lung cancer resistance to susceptibility at the Pas1 locus in Kras2LA2 mice. Nat Genet. 38, 926-930 92 Manenti, G., De Gregorio, L., Pilotti, S., Falvella, F. S., Incarbone, M., Ravagnani, F., Pierotti, M. A. and Dragani, T. A. (1997) Association of chromosome

16 12p genetic polymorphisms with lung adenocarcinoma risk and prognosis. Carcinogenesis. 18, 1917-1920 93 Yanagitani, N., Kohno, T., Sunaga, N., Kunitoh, H., Tamura, T., Tsuchiya, S., Saito, R. and Yokota, J. (2002) Localization of a human lung adenocarcinoma susceptibility locus, possibly syntenic to the mouse Pas1 locus, in the vicinity of the D12S1034 locus on chromosome 12p11.2-p12.1. Carcinogenesis. 23, 1177-1183 94 Falvella, F. S., Spinola, M., Manenti, G., Conti, B., Pastorino, U., Skaug, V., Haugen, A. and Dragani, T. A. (2007) Common polymorphisms in D12S1034 flanking genes RASSF8 and BHLHB3 are not associated with lung adenocarcinoma risk. Lung Cancer. 56, 1-7 95 Korkola, J. E., Houldsworth, J., Chadalavada, R. S., Olshen, A. B., Dobrzynski, D., Reuter, V. E., Bosl, G. J. and Chaganti, R. S. (2006) Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer Res. 66, 820-827 96 Weiser, K. C., Liu, B., Hansen, G. M., Skapura, D., Hentges, K. E., Yarlagadda, S., Morse Iii, H. C. and Justice, M. J. (2007) Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma. Mamm Genome. 18, 709-722 97 Eckfeldt, C. E., Mendenhall, E. M., Flynn, C. M., Wang, T. F., Pickart, M. A., Grindle, S. M., Ekker, S. C. and Verfaillie, C. M. (2005) Functional analysis of human hematopoietic stem cell gene expression using zebrafish. PLoS Biol. 3, e254 98 Jin, J., Smith, F. D., Stark, C., Wells, C. D., Fawcett, J. P., Kulkarni, S., Metalnikov, P., O'Donnell, P., Taylor, P., Taylor, L., Zougman, A., Woodgett, J. R., Langeberg, L. K., Scott, J. D. and Pawson, T. (2004) Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr Biol. 14, 1436-1450 99 Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M., Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S. L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C., Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S., Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P. and Vidal, M. (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature. 437, 1173-1178 100 Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E. and Eipper, B. A. (1996) Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme. J Biol Chem. 271, 28636-28640 101 Prigge, S. T., Mains, R. E., Eipper, B. A. and Amzel, L. M. (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci. 57, 1236-1259 102 Chen, L., Johnson, R. C. and Milgram, S. L. (1998) P-CIP1, a novel protein that interacts with the cytosolic domain of peptidylglycine alpha-amidating monooxygenase, is associated with endosomes. J Biol Chem. 273, 33524-33532 103 Rajagopal, C., Stone, K. L., Francone, V. P., Mains, R. E. and Eipper, B. A. (2009) Secretory granule to the nucleus: Role of a multiply phosphorylated intrinsically unstructured domain. J Biol Chem 104 Hesson, L. B., Dunwell, T. L., Cooper, W. N., Catchpoole, D., Brini, A. T., Chiaramonte, R., Griffiths, M., Chalmers, A. D., Maher, E. R. and Latif, F. (2009)

17 The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias. Mol Cancer. 8, 42 105 Reeves, N. and Posakony, J. W. (2005) Genetic programs activated by proneural proteins in the developing Drosophila PNS. Dev Cell. 8, 413-425 106 Nybakken, K., Vokes, S. A., Lin, T. Y., McMahon, A. P. and Perrimon, N. (2005) A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat Genet. 37, 1323-1332

Figure Legends

Figure 1. N-terminal RASSF proteins are structurally distinct from the classical RASSF proteins. The N-terminal RASSF proteins comprise a recently identified set of RA domain/ubiquitin fold containing proteins. Their domain architecture is distinct from the classical RASSF proteins suggesting they should be considered as a separate group. Sequences used for the domain analysis were; hsRASSF1A (NP_009113), hsRASSF2 (NP_055552), hsRASSF3 (NP_835463), hsRASSF4 (NP_114412), hsRASSF5/splice variant NORE1A (NP_872604), hsRASSF6B (NP_958834), hsRASSF7 (NP_003466), hsRASSF8 (NP_009142), P-CIP1/RASSF9 (AAD03250) and RASSF10 (NP_001073990, the short version described in [104]). Adapted from Figure 1 in [5].

Figure 2. The RA domains of the classical and N-terminal RASSF proteins are phylogenetically distinct. The phylogeny of the RA domains from RASSF1-10, ten other RA domains from nine other proteins (AF6 has two RA domains), and a yeast outlier was inferred. The analysis was carried out by profile aligning the RA domains to the alignment of RA domains from the SMART database, using ClustalW. Phylogenetic inference was then carried out using neighbor-joining, parsimony and maximum likelihood methods from PHYLIP3.67. There was some variation in the tree topologies, but with each method the RA domains from the classical and N-terminal proteins clustered in two well separated monophyletic groups. The tree here shows the maximum likelihood inference using the Jones-Taylor-Thornton model. RA domains from the following sequences were used; RASSF1A (NP_009113.3), RASSF2 (NP_055552.1), RASSF3 (NP_835463.1), RASSF4 (NP_114412.2), RASSF5 (NP_872604.1), RASSF6 (NP_803876.1), RASSF7 (NP_003466.1), RASSF8 (NP_009142.2), RASSF9 (AAD03250.1), RASSF10 (NP_001073990.1), STE50 (P25344), Rap1 (AF478469_1), A4beta (EAW86096.1), GRB10 (Q13322), MYOIXb (Q13459), GRB7 (NP_005301.2), DGKtheta (P52824), PhosC (Q5VWL4), AF-6 (BAA32485.1).

Figure 3. Emerging evidence suggests a possible link between the N-Terminal RASSF proteins and cancer progression. A summary of the evidence suggesting that the N-terminal RASSFs may play a role in tumourigenesis. Data consistent with potential anti-tumourigenic roles are indicated by red arrows, whilst evidence suggesting pro-tumourigenic roles are indicated by green arrows. Broken arrows highlight links which may be anti- or pro-tumourigenic. The cellular localisation is given where it is known. Full details and references are provided in the review text.

19 Tables

N-terminal Chromosome Alternative names Potential Potential RASSF Drosophila C. elegans member homologue homologue RASSF7 11p15.5 HRAS1 cluster1 (HRC1), C11orf13 CG5053 RASSF8 12p12.3 Human carcinoma associated HoJ- 1(HoJ-1), C12orf2 K05B2.2* RASSF9 12q21.31 peptidylglycine alpha-amidating CG13875* monooxygenase (PAM) C-terminal interactor 1 (P-CIP1), PAMC1 RASSF10 11p15.2 similar to peptidylglycine alpha- CG32150* amidating monooxygenase C- terminal interactor 1 Table 1. N-terminal RASSF family. The table summarises the nomenclature used for the N-terminal RASSF proteins. The genes marked with a * are not predicted to have an RA domain by the SMART database, however they do have sequence similarity over the RA domain region of the vertebrate N-terminal RASSF protein, thus can be considered as potential homologues.

Gene Tumour type Change Reference Pancreatic Up-regulated [74] + [75] adenocarcinoma RASSF7 Islet cell tumour Up-regulated [76] Endometrial carcinoma Up-regulated [77] Ovarian clear cell Amplified and [78] carcinoma up-regulated RASSF8 Lung adenocarcinoma Down-regulated [22] Male germ cell tumours Down-regulated [95] RASSF10 T cell acute lymphocytic Down-regulated by [104] leukaemia promoter methylation Table 2. Aberrant expression of the N-terminal RASSFs in cancer cells. The table presents a summary of manuscripts reporting aberrant expression of N- terminal RASSF members in cancer. There are other examples in the Oncomine data base (http://www.oncomine.org/), but we have only included those where we can find reference to the N-terminal member in the primary paper or supplementary material. It is important to note that these papers often use the alternative gene names described in Table 1.

Figure 3 Sherwood et al.